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ISSN : 1225-1577(Print)
ISSN : 2384-0900(Online)
The Korean Journal of Oral and Maxillofacial Pathology Vol.49 No.6 pp.151-163
DOI : https://doi.org/10.17779/KAOMP.2025.49.6.004

Spermidine Inhibits Mitochondrial Dysfunction through PPAR/PGC-1α Axis in Aging Salivary Gland

Nguyen Duy Phu, Sang-Gun Ahn*
Department of Pathology, School of Dentistry, Chosun University
* Correspondence: Sang-Gun Ahn, Ph.D., Department of Pathology, School of Dentistry, Chosun University, #309 Pilmun-Daero, Dong-gu, Gwangju, Republic of Korea, 61452 Tel: +82-62-230-6898 Email: ahnsg@chosun.ac.kr
December 1, 2025 December 15, 2025 December 26, 2025

Abstract


Aging is strongly linked to mitochondrial dysfunction, which disrupts energy metabolism and tissue homeostasis. The salivary gland is an energy-dependent organ, and mitochondrial decline contributes to age-related atrophy and impaired secretion. In this study, we investigated whether spermidine can restore mitochondrial function in aging salivary glands. In aging accelerated klotho deficient mouse, spermidine supplementation improved glandular histology, restoring acinar organization and reducing atrophy. Transcriptomic profiling revealed that spermidine induced extensive transcriptional reprogramming, characterized by the upregulation of mitochondrial metabolic pathways and suppression of inflammatory signaling. qRT-PCR and Western blot analyses confirmed increased expression of PPARγ mediated mitochondrial membrane potential such as PGC-1α, NRF1/2, and OXPHOS complex subunits. Furthermore, spermidine elevated mitochondria oxygen consumption rate, including basal respiration, ATP production, maximal respiration, and spare respiratory capacity. These results demonstrated that spermidine improves mitochondria respiratory capacity through activation of the PPAR–PGC-1α–NRF regulatory axis and may serve as a potential therapeutic strategy for restoring mitochondrial homeostasis and preserving salivary gland function during aging.


Aging , Salivary glands , Mitophagy , Spermidine , Mitochondria

Spermidin은 노화 타액선에서 PPAR/PGC-1α Axis을 통해 미토콘드리아 기능 장애 억제

응우옌 유이 푸, 안상건*
조선대학교 치과대학 병리학교실

초록

    Ⅰ. INTRODUCTION

    Aging is characterized by a progressive decline in physiological integrity, leading to reduced tissue resilience and increased susceptibility to disease (1,2). Among the hallmarks of aging, mitochondrial dysfunction plays a central role due to its impact on energy generation, redox balance, and cellular homeostasis. Accumulated damage to mitochondrial DNA, proteins, and membranes impairs oxidative phosphorylation, decreases ATP production, and increases reactive oxygen species (ROS), collectively driving the functional deterioration observed in aged tissues (3,4). Beyond biochemical decline, aging is also accompanied by disruption of mitochondrial quality-control networks—including mitochondrial dynamics, proteostasis, and mitophagy—all of which are essential for maintaining a healthy mitochondrial population (5,6).

    Mitochondrial homeostasis is associated with various mitochondrial biogenesis and bioenergetic pathways. Specifically, PGC-1α (peroxisome proliferator–activated receptor gamma coactivator-1α) acts as a master regulator of mitochondrial biogenesis by coactivating nuclear respiratory factors (NRF1 and NRF2) to induce genes involved in oxidative phosphorylation and mitochondrial maintenance. Reduced PGC-1α expression in aged tissues contributes to insufficient mitochondrial renewal and decreased respiratory chain integrity (7,8). Mitochondria exist as a dynamic network undergoing continuous cycles of fusion and fission that adapt to cellular stress and metabolic needs. Fusion, mediated by MFN1/MFN2 on the outer membrane and OPA1 on the inner membrane, enables mixing of mitochondrial contents and maintains bioenergetic capacity. Conversely, fission, coordinated by DRP1, FIS1, MFF, and MID49/51, facilitates mitochondrial redistribution and enables the segregation of damaged mitochondria for degradation (9). Age-associated imbalance in these processes results in excessive mitochondrial fragmentation, reduced membrane potential, inefficient ATP generation, and enhanced susceptibility to stress. Studies in various model organisms have demonstrated that perturbations in mitochondrial dynamics significantly affect lifespan and tissue homeostasis, underscoring their importance in aging (10,11).

    Spermidine, an endogenous polyamine, is increasingly recognized as a potent modulator of cellular homeostasis. Its levels decline with age, and supplementation has been shown to extend lifespan and improve mitochondrial function in multiple organisms (12,13). Spermidine enhances mitochondrial respiration, increases membrane potential, and stimulates pathways associated with mitochondrial biogenesis and metabolic regulation (7,14). Despite growing evidence of its protective effects in aging tissues, the impact of spermidine on mitochondrial function in aging salivary glands has not been fully elucidated.

    In this study, we investigated that spermidine can regulate the mitochondrial dysfunction in aging salivary glands. In primary salivary gland cells, spermidine induced mitochondrial membrane potential, oxygen consumption rate, membrane protein complexes, and the expression of major regulatory factors involved in mitochondrial biogenesis, including PPARγ, PGC-1α, and NRF1/2. Our findings indicated that spermidine enhances mitochondrial oxidative phosphorylation and overall mitochondrial performance in kl−/− salivary gland cells, providing mechanistic insight into its potential role as a therapeutic strategy against agerelated salivary gland dysfunction.

    Ⅱ. MATERIALS and METHODS

    1. Animals and treatment

    Klotho-deficient (kl-/-) mice were generated by mating pairs of heterozygous klotho mice (kl+/-) generously provided by Dr. Kuro‐o (University of Texas Southwestern, Dallas, TX, USA). At approximately 6 weeks of age, kl -/- mice were randomly assigned to treatment or control groups (n = 3 per group). The treatment group received spermidine (50 mg/kg body weight) via intraperitoneal injection once weekly for two consecutive weeks, as previously described. Spermidine was dissolved in sterile phosphate-buffered saline (PBS) and administered in a volume of 100 μL; control mice received an equal volume of vehicle (PBS) injection. One week after the second injection (total experimental duration of 3 weeks), mice were humanely sacrificed by cervical dislocation under anesthesia. Salivary glands (submandibular, sublingual, and parotid glands) were immediately harvested and either fixed for histology or snap-frozen in liquid nitrogen for molecular analyses. Experiments were performed under approved protocols of the animal research institute committee of Chosun university for the care and use of laboratory animals (CIACUC2024‐A0020).

    2. Histological analysis

    For morphological assessment, salivary gland tissues were fixed overnight in 10% neutral-buffered formalin at 4°C. Tissues were then dehydrated through graded ethanol, cleared in xylene, and embedded in paraffin. Paraffin blocks were sectioned at 4–5 μm thickness using a microtome. Sections were deparaffinized and rehydrated, then stained with hematoxylin and eosin (H&E) following standard protocols. After staining, sections were rinsed, dehydrated, cleared, and mounted with coverslips. Histological morphology of the salivary glands was examined under a light microscope at appropriate magnifications. Representative images were captured using a digital slide scanner or camera attached to the microscope. The glandular architecture, acinar cell morphology, and presence of any age-related histopathological changes were qualitatively assessed by a blinded observer.

    3. RNA seq analysis

    Total RNA was extracted from mouse salivary gland tissues using TRIzol Reagent (Invitrogen), and quality was assessed with the Agilent 4200 TapeStation System. RNA samples were sent to an external sequencing facility for library preparation using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD (Lexogen). Sequencing was performed on an Illumina NextSeq 2000 platform to generate single-end 100 bp reads. Raw sequencing reads were preprocessed using fastp v0.23.1 to remove adapter sequences and low-quality reads. The filtered reads were then aligned to the reference genome using STAR v2.7.10b. The read counts were quantified using feature counts v2.0.6. For differential gene expression analysis, edgeR package v3.42.4 was used, applying TMM-normalized log2(CPM+1) transformation to normalize the data. Functional enrichment analysis was performed using clusterProfiler and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http:// www.genome.jp/kegg/). Full visual summaries of differential expression and pathway including heatmaps, volcano plots, pie charts of biological processes, and KEGG pathway enrichment bar graphs and networks connectivity. Fisher's exact test was used to identify the significant functions and pathways represented within the respective gene sets.

    4. Cell culture and treatment

    Mutant primary salivary gland cells (PSGCs kl-/-) were generated as previously described (15). The PSGCs kl-/- cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Welgene Inc., Gyeongsanbuk-do, Korea) containing 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained in a 5% CO2 humidified atmosphere at 37° C. Once cells reached 70–80% confluence, they were used for experiments. For in vitro spermidine treatment, PSGCs kl -/- were seeded in 6-well plates and allowed to adhere overnight. The next day, cells were treated with spermidine at final concentrations of 0 μM (vehicle control), 20 μM, or 50 μM for 24 h. Spermidine stock solution (10 mM) was prepared in sterile water and diluted into culture medium to achieve the desired concentrations. Control cells received an equal volume of water (vehicle). After 24 h, cells were observed for viability and then harvested for analyses. All treatments were performed in triplicate wells for each condition. Following treatment, cells were either lysed for protein extraction or subjected to the mitochondrial membrane potential assay described below.

    5. Western blot analysis

    Salivary gland tissues and cultured PSGCs were lysed using radioimmunoprecipitation assay (RIPA) buffer (Biosesang, Seongnam, Korea) containing a protease inhibitor cocktail (1 μg/mL) and phosphatase inhibitor (1 μg/mL). Then, equal amount of protein (20 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS– PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Burlington, NJ, USA). The membranes were blocked with 5% skim milk for 2 h and then incubated overnight at 4° C with primary antibodies (1:1000, diluted in TBST). All primary antibodies were diluted in TBS + 0.1% Tween 20 (TBST) at a 1:1000 ratio. The primary antibodies included anti-PPARγ (sc-7273; Santa Cruz), anti-NDUFV2 (sc-271620; Santa Cruz), anti-SHDB (sc-271548; Santa Cruz), anti-NRF1 (sc-101102; Santa Cruz) , anti-NRF2 (sc-365949; Santa Cruz), anti-UQCRC2 (sc-390378; Santa Cruz), anti- MTCO1 (E2I2R; Cell Signaling) and anti-PGC1α (66369-1-Ig; Proteintech). On the next day, the membranes were washed 3 x 5 minutes with TBST and then incubated with appropriate secondary antibodies conjugated to HRP (Promega, Madison, WI, USA) for 1 h at room temperature. The protein signal was visualized by Immobilon® Western Chemiluminescent HRP Substrate reagents (Merck Millipore, Burlington, NJ, USA) through a luminescence image analyzer (LAS-1000, Fujifilm, Tokyo, Japan).

    6. Quantitative reverse transcription polymerase chain reaction (qRT–PCR) analysis

    Total RNA was extracted from frozen submandibular gland tissues of control and spermidine-treated kl -/- mice. Tissues (~30 mg) were homogenized in TRIzol reagent (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. RNA quality was determined by OD280/260 to ensure that the RNA template had a 260/280 ratio between 1.8-2.0. Reverse transcription was performed on 1 μg of total RNA using oligo dT primers and M-MLV Reverse Transcriptase (Invitrogen) in a final volume of 20 μL for 5 min at 65° C followed by 1 hr at 37° C. Quantitative reverse transcription PCR was performed using the GoTaq® 1-Step RT–qPCR System kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The primer sets used are shown in Table 1. The relative expression level of target genes is represented by the 2-ΔΔCt value.

    7. Mitochondrial membrane potential assay (JC-1)

    Mitochondrial membrane potential in PSGCs kl-/- was assessed using the JC-1 dye (Dojindo, Japan). After treatment with 20 μM spermidine for 24 h, cells grown on chamber slides were incubated with JC-1 working solution (2 μM final concentration) at 37 for 20 minutes in the dark. Following incubation, cells were washed with warm HBSS to remove excess dye. Live cell imaging was performed using a confocal microscope (Nikon Corporation, Tokyo, Japan). Red fluorescence (JC-1 aggregates) was detected with excitation at 540 nm and emission at 590 nm, while green fluorescence (JC-1 monomers) was measured with excitation at 485 nm and emission at 535 nm. The mitochondrial membrane potential was quantified by calculating the ratio of red to green fluorescence intensity per field using ImageJ software. A higher red/green ratio indicates higher mitochondrial polarization. Each experimental condition was analyzed in at least three independent wells.

    8. Oxygen consumption rate assay

    The oxygen consumption rate (OCR) of the kl-/- PSGCs was determined by a Seahorse XF HS Mini Analyzer from Agilent Technologies using an XF Cell Mito Stress Test kit (Agilent Technologies) according to the manufacturer’s instructions. Briefly, cells were plated with 3 replicates into a Seahorse XFp Cell Culture Miniplate (Agilent Technologies) at a density of 20,000 cells/well, followed by treatment with 20 μM spermidine for 24 h. These cells were then used for the Seahorse assay. First, the cells were incubated in a CO2-free incubator for 1 h before measurement, after which the baseline OCR was recorded. Oligomycin (1 μM), FCCP (0.5 μM) and rotenone/antimycin A (1 μM) were subsequently added to reveal the key metabolic function parameters. The basal respiration, ATP production, maximal respiration and spare capacity were calculated.

    9. Statistical analysis

    The data are presented as the means ± standard error of the means (SEMs). Statistical analyses and graphical representations were conducted using GraphPad Prism 10 software. To determine the significant difference between the control and experimental groups, Student’s t-test was applied. All the data were obtained from three independent experiments. Statistical significance was established at * p < 0.05, ** p < 0.01, *** p < 0.001.

    Ⅲ. RESULTS

    1. Spermidine preserves salivary gland histological property in kl-/- mice

    To determine whether spermidine can attenuate age-associated salivary gland degeneration in kl-/- mice, spermidine (50 mg/kg, i.p.) was administered on 1 and 7 days, and all major salivary glands were collected on day 14 (Fig. 1A). In untreated kl-/- mice, H&E-stained sections of the parotid, sublingual, and submandibular glands showed pronounced age-related pathology. The normal lobular organization was disrupted, with diffuse acinar loss, reduced numbers of both mucous and serous acini, and extensive areas of glandular atrophy. Acinar cells frequently appeared shrunken and disorganized (Fig. 1B). By contrast, spermidine-treated kl-/- mice displayed a substantial preservation of glandular morphology. The acinar cells in sublingual and submandibular glands appeared more densely packed and better organized, with an increased number of intact mucous and serous acini compared with untreated controls (Fig. 1C).

    2. RNA-seq of salivary gland in spermidine treated kl-/- mice

    To examine the molecular mechanism of spermidine, RNA seq analysis was performed on submandibular glands from spermidine-treated kl-/- mice. The clustering of differentially expressed genes (DEGs) revealed a clear separation between the two groups by heat map (Fig. 2A). A volcano plot further demonstrated that numerous genes were significantly altered by spermidine treatment (Fig. 2B). Genes associated with mitochondrial lipid metabolism and mitochondrial function, including Ucp1, Cidea, Fabp4, Pck1, and Lpl, were strongly upregulated, whereas multiple pro-inflammatory genes such as Trem2, Cxcl family members, and IL2 were downregulated. Gene ontology (GO) analysis showed that DEGs were enriched in pathways related to mitochondrial biogenesis, secretory function, DNA repair, immune regulation, and cell differentiation (Fig. 2C). Functional grouping of these categories revealed a prominent enrichment of genes associated with mitochondrial biogenesis, mitophagy-related processes, and salivary gland secretion, whereas genes linked to inflammation and immune responses were significantly suppressed (Fig. 2D). Consistent with these observations, KEGG pathway analysis demonstrated activation of multiple metabolic programs, including the PPAR signaling pathway, fatty acid degradation, TCA cycle, oxidative phosphorylation, and AMPK signaling (Fig. 2E). In contrast, pathways associated with NF-κB signaling, cytokine–cytokine receptor interaction, B-cell receptor signaling, and JAK–STAT signaling were significantly downregulated (Fig. 2F).

    3. Spermidine upregulates genes involved in lipid metabolism and mitochondrial β-oxidation

    To validate the RNA-seq results, quantitative real-time RT –PCR was performed on submandibular gland RNA from spermidine-treated kl-/- mice. Consistent with the RNA seq data, spermidine treatment significantly increased mRNA levels of key metabolic genes involved in lipid transport genes (Lpl, Fabp4), thermogenic factors (Cidea, Ucp1), and ion-transport genes (Atp1a2). In particular, Lpl and Fabp4, which mediate lipid uptake and intracellular fatty acid transport, were significantly upregulated in the spermidine-treated group compared with controls. Expression of Cidea and Ucp1, typically associated with thermogenic and energy-dissipating processes, was also significantly elevated, suggesting that spermidine promotes the basal metabolic rate in kl-/- salivary gland. Furthermore, Atp1a2, encoding the Na⁺ /K⁺-ATPase α2 subunit essential for maintaining ion gradients and fluid secretion in salivary acinar cells, was significantly increased following spermidine administration (Fig. 3A). In addition, spermidine also induced upregulation of genes involved in mitochondrial fatty acid β-oxidation. mRNA of Cpt1a and Cpt2, which mediate fatty acid import into mitochondria, were significantly elevated in spermidine- treated glands. Downstream β-oxidation enzymes, including Acadvl (very-long-chain acyl-CoA dehydrogenase), Acaa2 (acetyl-CoA acyltransferase 2), and Echs1 (enoyl-CoA hydratase 1), were also importantly upregulated.

    4. Spermidine activates mitochondria regulatory signaling and increases expression of OXPHOS complexes

    To determine whether spermidine influences mitochondrial regulatory pathways, we next evaluated the expression of regulators of mitochondrial biogenesis and key components of the mitochondria electron transport chain. First, qRT –PCR analysis showed that spermidine significantly increased mRNA expression of PPARγ, PPARα, PGC-1α, NRF1, and NRF2 compared with untreated kl-/- mice (Fig. 4A). Western blot analysis further confirmed this expression. In salivary gland lysates, spermidine-treated kl−/− mice displayed higher protein expression of PPARγ, PGC-1α, NRF1, and NRF2 relative to controls (Fig. 4B). A similar pattern was observed in aged primary salivary gland cell (PSGCs kl-/-) exposed to increasing concentrations of spermidine (0, 20, 50 μM) (Fig. 4C). In addition, in salivary gland tissue, spermidine significantly increased mRNA levels of representative OXPHOS genes from Complex I (Ndufv2), Complex II (Sdhb), Complex III (Uqcrc2), and Complex IV (Cox8b) (Fig. 4D). Western blot analysis of salivary gland lysates confirmed that protein levels of these complex subunits were also upregulated in spermidine-treated kl-/- mice (Fig. 4E).

    5. Spermidine induces mitochondrial membrane potential and ATP production in kl-/- PSGCs

    To directly assess mitochondrial function, we examined mitochondrial membrane potential and oxygen consumption in kl-/- PSGCs treated with spermidine. JC-1 staining revealed that kl-/- PSGCs exhibited predominantly green fluorescence, indicative of low mitochondrial membrane potential and a depolarized mitochondrial state. In contrast, spermidine-treated cells displayed intense red fluorescence corresponding to JC-1 aggregates (Fig. 5A). Quantification of the red/green fluorescence ratio also significantly increased the mitochondrial membrane potential in the spermidine- treated cells compared with untreated controls (Fig. 5B). Mitochondrial respiratory function was further evaluated using a Seahorse XF HS Mini Analyzer. As shown in the oxygen consumption rate (OCR), spermidine-treated PSGCs exhibited higher basal respiration than control cells and displayed greater responsiveness to sequential injections of oligomycin, FCCP, and rotenone/antimycin A (Fig. 5C). Calculation of bioenergetic parameters revealed that spermidine significantly enhanced basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity compared with vehicle-treated cells (Fig. 5D).

    6. Spermidine regulates OXPHOS pathways through PPAR/PGC-1 signaling

    To better understand how spermidine-responsive genes are functionally interconnected, a protein–protein interaction (PPI) network was constructed based on the RNA-seq dataset. The resulting network revealed strong connectivity among genes belonging to PPAR signaling, mitochondrial biogenesis regulation, and oxidative phosphorylation (Fig. 6). PPAR/PGC-1α tightly linked OXPHOS components and mitochondrial biogenesis regulators, indicating that these pathways are showed in spermidine mediated mitochondria OXPHOS pathways.

    Ⅳ. DISCUSSION

    Aging is accompanied by progressive mitochondrial impairment, metabolic insufficiency, and chronic activation of inflammatory pathways, all of which contribute to the functional deterioration of salivary glands (16-19). In kl-/- mice, these age-associated alterations appear as acinar atrophy, disorganization of glandular structures, and profound energy metabolism decline by mitochondria damage. In this study, we investigated to determine whether spermidine could inhibit these pathological changes. Salivary glands in kl-/- mice showed the degenerative features, including an atrophy of acinar organization and extensive loss of secretory cells, reflecting severe structural change. We found that spermidine induces number of intact mucous and serous acini in kl-/- mice. These histological observations indicated that spermidine treatment is sufficient to improve functional degeneration in kl-/- salivary glands. Using RNA-seq analysis, we examined the differentially expressed genes in spermidine treated salivary glands. RNA sequencing revealed a pronounced shift in gene expression patterns, with treated and untreated tissues forming clearly distinct clusters. Genes governing mitochondrial metabolism, oxidative phosphorylation, fatty acid utilization, and TCA cycle activity were strongly upregulated, indicating a metabolic reactivation that supports enhanced cellular energy production. At the same time, inflammatory and immune-related transcripts—particularly those involved in NF-κB signaling and cytokine interactions— were significantly downregulated. This combination of metabolic activation and inflammatory suppression reflects an inhibition of two major hallmarks of aging such as bioenergetic decline and chronic inflammation..

    In this study, we found that spermidine induces the reactivation of the PPAR–PGC-1α–NRF axis, a key regulatory pathway that manage mitochondrial biogenesis and oxidative metabolism. In kl-/- glands, this axis is importantly impaired, which contributes to deficits in lipid metabolism, reduced mitochondria regulator, and diminished OXPHOS capacity (7,20-22). Spermidine treatment restored the expression of PPARγ, PPARα, PGC-1α, NRF1, and NRF2. In parallel, genes encoding components of the OXPHOS complexes were upregulated, suggesting that increased transcriptional drive translates into enhanced mitochondrial structural and functional capacity. These mechanism changes by spermidine were comfired through functional assays of mitochondrial activity. Spermidine significantly increased mitochondrial membrane potential in kl-/- PSGCs, demonstrating improved electron transport chain performance. Additionally, measurements of oxygen consumption showed substantial improvements in basal respiration and ATP production. Together, these functional improvements suggest that spermidine not only activates mitochondrial gene expression but also restores the physiological ability of mitochondria to generate energy efficiently. In addition, the network analysis integrating differentially expressed genes also showed strong connectivity among PPAR signaling components, mitochondrial biogenesis regulators, and OXPHOS complexes. Such coordinated remodeling aligns with previous studies in other tissues where spermidine has been shown to promote mitochondrial activity and extend cellular longevity (23,24).

    Although the present study showed the roles of spermidine on the mitochondrial and metabolic mechanisms in kl-/- salivary gland, there is several limitations. Spermidine treatment was relatively short, preventing evaluation of long-term efficacy, safety, or the durability of functional recovery. Importantly, direct functional measurements such as saliva secretion in spermidine treated mouse were not performed in spermidine treated aging mouse. Therefore, in future study, we will investigate long-term effects of spermidine to determine whether the functional and metabolic benefits observed kl-/- aging animal models. In addition, we will extend these findings by examining spermidine’s effects in naturally aged animal models to confirm its therapeutic potential.

    Our results demonstrated that spermidine protective the aging salivary gland by restoring mitochondria functional signaling. These findings provide evidence that spermidine may act as a mitochondria-targeted metabolic modulator and serve as a promising therapeutic strategy for preserving salivary gland health during aging.

    AKNOWLEDGEMENTS

    This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2023-00222390).

    Figure

    KAOMP-49-6-151_F1.jpg

    Histological features of the salivary gland in kl-/- mice

    (A) Experimental scheme showing spermidine administration (50 mg/kg, intraperitoneal) on days 1 and 7 and tissue harvest on day 14. (B) Representative H&E-stained sections of parotid, sublingual, and submandibular glands in kl-/- mice (C) H&E Staining of sublingual, and submandibular glands in spermidine treated kl-/- mice (×200 and ×400 magnification).

    KAOMP-49-6-151_F2.jpg

    RNA-seq analysis in spermidine treated kl-/- salivary glands.

    (A) Heatmap clustering of differentially expressed genes (DEGs) between control and spermidine-treated kl-/- submandibular glands (B) Volcano plot highlighting significantly up- and downregulated genes (p < 0.05). (C–D) GO enrichment analyses showing altered genes involved in biological process. The gene expression ratio (≥2‐fold) was evaluated from gene expression profiles in the submandibular glands of kl-/- and spermidine-treated kl-/- mice. (E) Functional pathway of the up regulated genes identified from comparative RNA-seq analysis of spermidine-treated kl-/- submandibular glands via KEGG pathway enrichment analysis. (F) KEGG analysis of the down-regulated genes in spermidine-treated glands.

    KAOMP-49-6-151_F3.jpg

    Spermidine induces lipid metabolism and mitochondrial β-oxidation gene expression.

    Validation of differentially expressed genes in kl-/- and spermidine-treated kl-/- mice. Total RNAs were extracted from submandibular gland tissues isolated from individual mice. cDNA was synthesized by reverse transcription‐polymerase chain reaction (RT‐PCR). mRNA levels were normalized to GAPDH. (A) qRT-PCR validation of lipid transport and thermogenic genes (Lpl, Fabp4, Cidea, Ucp1) and ion-transport–related Atp1a2 in spermidine-treated kl−/− glands. (B) qRT-PCR validation of β-oxidation–related genes (Cpt1a, Cpt2, Acadvl, Acaa2, and Echs1). The data are reported as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.001.

    KAOMP-49-6-151_F4.jpg

    Spermidine activates mitochondrial biogenesis regulators and OXPHOS complexes.

    (A) Total RNAs were extracted from submandibular gland of spermidine-treated kl-/- mice. Mitochondrial biogenesis regulators (PPARγ, PPARα, PGC-1α, NRF1, and NRF2) were measured by qRT-PCR analysis. (B) Expression of PPARγ, PPARα, PGC-1α, NRF1, and NRF2 protein in submandibular gland of spermidine-treated kl-/- mice. The total protein was extracted, and PPARγ, PPARα, PGC-1α, NRF1, and NRF2 protein levels were measured by Western blot, respectively. Actin was used as a loading control. (C) Western blot analysis of PPAR γ, PGC-1α, NRF1, and NRF2 protein levels in kl -/- PSGCs treated with 20 or 50 μM spermidine. (D) Spermidine enhances mRNA levels of mitochondria OXPHOS complexs: NDUFV2, SDHB, UQCRC2, and COX8b/MT-CO1. (E) Western blot analysis of mitochondria OXPHOS complex proteins in salivary gland from kl-/- and spermidine-treated kl -/- mice. *p < 0.05; **p < 0.01; ***p < 0.001.

    KAOMP-49-6-151_F5.jpg

    Spermidine induces mitochondrial membrane potential and respiratory function in kl-/- PSGCs.

    (A) Mitochondrial membrane potential detected by JC-1 fluorescence staining in spermidine treated kl -/- PSGCs, showing green fluorescence (low mitochondrial membrane potential) and red fluorescence (high). (B) Bar graph quantifying the red/green fluorescence intensity ratio shows a significant increase in the spermidine-treated kl-/- salivary gland compared to kl-/- mice. **p < 0.01. (C) The oxygen consumption rate (OCR) was measured in kl-/- PSGCs following the addition of spermidine (20 μM) for 24 h. (D) Quantification of bioenergetic parameters showing increased basal respiration, ATP production, maximal respiration, and spare respiratory capacity in spermidine-treated cells. Values represent mean ± SD. *p < 0.05.

    KAOMP-49-6-151_F6.jpg

    Network-level integration of spermidine-responsive mitochondrial pathways in kl-/- salivary glands.

    Network connectivity of differentially regulated genes in the salivary glands of spermidine treated kl-/- mice. Ingenuity Pathway Analysis was applied to genes showing significant dysregulation, with filtering on their relative distance from the mean ratio of the population. The first main pathway that appeared to be differentially expressed was PPAR γ signaling, mitochondrial biogenesis factors, and oxidative phosphorylation genes upregulated by spermidine.

    Table

    Primer sequences for qRT-PCR validation

    Reference

    1. López-Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G: The hallmarks of aging. Cell 2013;153:1194-217.
    2. Toan NK, Ahn SG: Aging-related metabolic dysfunction in the salivary gland: a review of the literature. International Journal of Molecular Sciences 2021;22:5835.
    3. Haas RH. Mitochondrial dysfunction in aging and diseases of aging. Biology 2019;8:48.
    4. Zhang X, Gao Y, Zhang S, Wang Y, Pei X, Chen Y, et al.: Mitochondrial dysfunction in the regulation of aging and aging-related diseases. Cell Communication and Signaling 2025;23:290.
    5. Figge MT, Osiewacz HD, Reichert AS. Quality control of mitochondria during aging: Is there a good and a bad side of mitochondrial dynamics? BioEssays 2013;35:314–22.
    6. Diniz LP, Araujo AP, Carvalho CF, Matias I, de Sá Hayashide L, Marques M, et al.: Accumulation of damaged mitochondria in aging astrocytes due to mitophagy dysfunction: Implications for susceptibility to mitochondrial stress. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 2024; 1870:167470.
    7. Wang J, Li S, Wang J, Wu F, Chen Y, Zhang H, et al. Spermidine alleviates cardiac aging by improving mitochondrial biogenesis and function. Aging (Albany, NY) 2020;12:650–71.
    8. Gorgori-Gonzalez A, Soto-Rodriguez S, Tamayo-Torres E, Garcia-Dominguez E, Sebastia V, Gambini J, et al,: Leveraging mitochondrial stress to improve healthy aging. Sports Medicine and Health Science 2025.
    9. Youle RJ, van der Bliek AM: Mitochondrial fission, fusion, and stress. Science 2012;337:1062–5.
    10. Chen W, Zhao H, Li Y: Mitochondrial dynamics in health and disease: mechanisms and potential targets. Signal transduction and targeted therapy 2023;8(1):333.
    11. Sharma A, Smith HJ, Yao P, Mair WB: Causal roles of mitochondrial dynamics in longevity and healthy aging. EMBO reports 2019;20:e48395.
    12. Nishimura K, Shiina R, Kashiwagi K, Igarashi:. Decrease in Polyamines with Aging and Their Ingestion from Food and Drink. J Biochem (Tokyo) 2006;139:81–90.
    13. Madeo F, Bauer MA, Carmona-Gutierrez D, Kroemer G: Spermidine: a physiological autophagy inducer acting as an anti-aging vitamin in humans? Autophagy 2018;15:165–8.
    14. Szabo L, Lejri I, Grimm A, Eckert A: Spermidine Enhances Mitochondrial Bioenergetics in Young and Aged Human- Induced Pluripotent Stem Cell-Derived Neurons. Antioxidants 2024;13:1482.
    15. Tai NC, Kim SA, Ahn SG: Soluble klotho regulates the function of salivary glands by activating KLF4 pathways. Aging (Albany NY) 2019;11:8254–69.
    16. Li N, Ye Y, Wu Y, Li L, Hu J, Luo D, Li Y, Yang J, Gao Y, Hai W, Xie Y, Jiang L: Alterations in histology of the aging salivary gland and correlation with the glandular inflammatory microenvironment. iScience 2023;26(5):106571.
    17. Chen Z, Mao QY, Zhang JY, Wu YX, Shan XF, Geng Y, Fan JY, Cai ZG, Xiang RL: Cellular Senescence Contributes to the Dysfunction of Tight Junctions in Submandibular Glands of Aging Mice. Aging Cell 2025; 24(5):e14470.
    18. Sutarjo FN, Rinthani MF, Brahmanikanya GL, Parmadiati AE, Radhitia D, Mahdani FY: Common precipitating factors of xerostomia in elderly. Journal of Health and Allied Sciences NU 2024;14:011-6.
    19. Proctor GB, Shaalan AM: Disease-induced changes in salivary gland function and the composition of saliva. Journal of dental research 2021;100:1201-9.
    20. Kwon SM, Kim SA, Yoon JH, Yook JI, Ahn SG: Global analysis of gene expression profiles in the submandibular salivary gland of klotho knockout mice. J Cell Physiol 2018;233:3282 –94.
    21. Qian L, Zhu Y, Deng C, Liang Z, Chen J, Chen Y, et al.: Peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) family in physiological and pathophysiological process and diseases. Signal Transduct Target Ther 2024;9:50.
    22. Finck BN, Kelly DP: Peroxisome Proliferator-Activated Receptor γ Coactivator-1 (PGC-1): Regulatory Cascade in Cardiac Physiology and Disease. Circulation 2007;115:2540-8.
    23. Ni YQ, Liu YS: New Insights into the Roles and Mechanisms of Spermidine in Aging and Age-Related Diseases. Aging Dis. 2021;12(8):1948-1963.
    24. Morselli E, Galluzzi L, Kepp O, Criollo A, Maiuri MC, Tavernarakis N, Madeo F, Kroemer G: Autophagy mediates pharmacological lifespan extension by spermidine and resveratrol. Aging (Albany NY) 2009;1(12):961-70.
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